Compositions and methods for TIGR genotyping assays

ABSTRACT

Methods and compositions are described for use in the rapid and simultaneous screening of one or more samples for one or more polymorphisms in the TIGR gene. The methods and compositions of the present invention can be used to rapidly determine if polymorphisms in a gene encoding the TIGR protein are present in the genome of a subject. Identifying which polymorphisms are present in an individual can permit the diagnosis or prediction of the risk of glaucoma in the subject.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.10/017,870, now issued U.S. Pat. No. 6,727,354, filed Dec. 12, 2001,incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the field of diagnostic assays and assays foridentifying patients at risk for development of glaucoma.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Glaucoma is the second most common cause of blindness in the UnitedStates. It is estimated that some two million Americans have glaucoma,with half of those suffering unaware of the presence of the disease.Primary open-angle glaucoma (“POAG”) is the most common form ofglaucoma, accounting for some 60 to 70% of all glaucomas.

POAG is characterized by obstruction of the normal aqueous outflow offluids through the trabecular meshwork, canal of Schlemm, intrascleralchannels, and episcleral and conjunctival veins. In open-angle glaucoma,this obstruction exists despite an angle that appears open. Generally, apatient that has not been otherwise diagnosed as having glaucoma firstbecomes aware of the disease due to losses in the visual field. By thispoint, the degree of optic nerve atrophy resulting from the disease maybe quite severe, and is irreversible. Thus, early diagnosis andtreatment play a key role in patient management.

Several risk factors have been identified as being related to POAG,including elevated intraocular pressure (“IOP,” 50% of patients presentwith an IOP of <22 mm Hg), increased age (POAG is 6× more common inpersons >60 years of age), a family history of the disease (a 15×increased chance of developing glaucoma), race (African Americans are atan increased risk for more serious disease), diabetes, hypertension,myopia, and the use of corticosteroids.

While elevated IOP is a primary risk factor for development of POAG,about ⅙ of patients exhibit an IOP within the normal range.Additionally, there is currently no reliable method for predicting whichpatients presenting with elevated IOP will progress to POAG. Recently,the relationship of the trabecular meshwork-inducible glucocorticoidresponse (“TIGR”) gene and protein have been studied for possibleassociations with glaucoma and related diseases. See, e.g., U.S. Pat.Nos. 5,861,497, 5,916,778, 5,925,748, and 6,248,867; Morissette et al.,Nat. Genet. 19: 319-21 (1998); Brezin et al., Am. J. Med. Genet. 76:438-45 (1998); Shimizu et al., Am. J. Ophthalmol. 130: 165-77 (2000);Nguyen et al., J. Biol. Chem. 273: 6341-50 (1998); and Lindblad-Toh etal., Nat. Genet. 24: 381-6 (2000). Each publication and patent in theforegoing section is hereby incorporated by reference in its entirety,including all tables, figures, and claims.

SUMMARY OF THE INVENTION

The present invention is drawn to methods and compositions for thescreening of samples for one or more TIGR polymorphisms. The sample canbe a biological sample, such as a sample from a subject. The inventioncan be used to determine which of a plurality of TIGR polymorphisms arepresent in the genome of a subject. Preferably, a plurality of differentsamples are assayed, each in its own individual reaction mixture, and/orseveral different polymorphisms in the TIGR gene are assayed in thatsingle reaction mixture. Thus, a plurality of samples may besimultaneously assayed for several different TIGR polymorphisms in asingle cycle (batch run) of the assay.

In a first aspect, the invention provides methods of testing for thepresence of one or more polymorphisms of a TIGR gene, in one or moresamples comprising TIGR nucleic acids, by generating a labeled nucleicacid that provides a means of identifying a particular polymorphism,thus distinguishing that polymorphism from other polymorphisms thatmight be present in the same gene. The particular polymorphism may beidentified, for example, by determining both the length of the labelednucleic acid and the identity of a distinctively labeled nucleotideincorporated at an end of the nucleic acid.

In preferred embodiments, these methods comprise one or more of thefollowing steps: (a) preparing a reaction mixture that contains (i)sample nucleic acid suspected of containing a TIGR nucleic acidsequence, (ii) a nucleic acid polymerase, (iii) one or more extensionprimers, wherein the extension primers comprise nucleotide sequencesthat terminate at positions located one nucleotide 3′ from the positionsof one or more preselected polymorphism(s) of interest, and (iv) one ormore labeled dideoxynulceotide triphosphates, or ddNTPs; (b) incubatingthe reaction mixture under conditions such that extension primers thathybridize to the TIGR nucleic acids are labeled by addition of one ofthe ddNTPs comprising a label to the 3′-end of the detection primer, inorder to generate one or more labeled oligonucleotides; and (c)detecting a signal from the labeled oligonucleotides. The presence of aspecific polymorphism can be identified by the presence of a distinctivesignal at a position in the sequence of the extended nucleic acid.

In certain embodiments, TIGR nucleic acid obtained from a sample isamplified to provide an amount of TIGR nucleic acid sufficient forprimer extension to determine the presence or absence of one or morepolymorphic forms of TIGR in the original sample. While the exemplarymethods described hereinafter relate to amplification using thepolymerase chain reaction (“PCR”), numerous other methods are known inthe art for amplification of nucleic acids (e.g., isothermal methods,rolling circle methods, etc.). The skilled artisan will understand thatthese other methods may be used either in place of, or together with,PCR methods.

The phrase “TIGR nucleic acid” refers to any nucleic acid containingsequences directly associated with production of the TIGR protein,including TIGR genomic DNA, TIGR-encoding hnRNA, mature TIGR-encodingmRNA, or amplification products thereof. Any TIGR nucleic acid may bethe subject of the methods described herein. In certain embodiments forexample, a TIGR RNA may be reverse transcribed into DNA, and the DNAsubjected to the analysis methods described hereinafter. In preferredembodiments, the methods are applied to TIGR gene sequences.

The phrases “TIGR gene sequences,” “TIGR genomic DNA,” and “TIGR gene”as used herein refer to the nucleic acid unit present in the genome ofan animal, preferably a human, encoding the TIGR protein, and includesboth the TIGR coding sequence and the upstream enhancer and promoterregions operably associated with the TIGR coding sequence in the genome.

The term “biological sample” as used herein refers to a sample obtainedfrom a biological source, e.g., an organism, cell culture, tissuesample, etc. A biological sample can, by way of non-limiting example,consist of or comprise blood, sera, urine, feces, epidermal sample, skinsample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrowsample and/or chorionic villi. Convenient biological samples may beobtained by, for example, scraping cells from the surface of the buccalcavity.

The term “subject” as used herein refers to any eukaryotic organism.Preferred subjects are fungi, invertebrates, insects, arachnids, fish,amphibians, reptiles, birds, marsupials and mammals. A mammal can be acat, dog, cow, pig, horse, ox, elephant, simian. Most preferred subjectsare humans. A subject can be a patient, which refers to a humanpresenting to a medical provider for diagnosis or treatment of adisease. The term “animals” includes prenatal forms of animals, such asfetuses.

As used herein, a “plurality of samples” refers to at least two.Preferably, a plurality refers to a relatively large number of samples.A plurality of samples is from about 5 to about 500 samples, preferablyabout 25 to about 200 samples, most preferably from about 50 to about200 samples. Samples that are processed in a single batch run of themethod of the invention are usually prepared in plates having 24, 48,96, 144, or 192 wells. The term “samples” includes samples per se aswell as controls, standards, etc. that are included in a batch run.

A “preselected TIGR polymorphism” is a TIGR nucleic acid sequence thathas been selected for testing by the methods of the invention. Examplesof preselected TIGR polymorphisms include wild type TIGR, and singlebase polymorphisms referred to herein as MT-1 (a promoter mutation), andT377M, E423K, and N480K (all exon 3 mutations). The MT-1 mutationreplaces C with G at position 906 in the promoter sequence of TIGR (FIG.2). The T377M mutation replaces C with T at position 468 in the TIGRcoding sequence, leading to a substitution of Thr with Met in the TIGRprotein; the N480K mutation replaces C with A at position 778 in theTIGR coding sequence, leading to a substitution of Asn with Lys in theTIGR protein; and the K423E mutation replaces A with G at position 605in the TIGR coding sequence, leading to a substitution of Lys with Gluin the TIGR protein (FIG. 1).

The assays described herein can be used to rapidly determine which of aselected group of polymorphic TIGR nucleic acid forms are present in asample comprising TIGR nucleic acid. By “rapid” it is meant that thelength of time that is taken to carry out a single batch run of theassay, from the moment a reaction mixture comprising nucleic acid isprepared to the moment a signal can be detected, is from about 1 secondto about 10, 15 or 30 seconds, about 1, 5, 10 or 30 minute(s), or 1, 3,5, 8, 24 or 48 hour(s). When samples are from multiple subjects, theassays can be used to determine the TIGR genotype of each subject.

By “distinctively labeled”, it is meant that each type of member of aset is labeled with a label that can be distinguished from the labelsused for other members of the set. For example, in a set ofdistinctively labeled nucleotides (e.g., dideoxy NTPs, or ddNPTs), eachtype of “N” (nucleotide) is labeled with a label that can bedistinguished from the other types of labels. Thus, for example, if fourlabels designated 1, 2, 3, and 4 are used to label the four types ofddNTPs, each ddATP molecule is labeled with label “*1”, each ddTTPmolecule is labeled with label “*2”, each ddCTP molecule is labeled withlabel “*3”, and each ddGTP molecule is labeled with label “*4”. In someaspects of the invention, the distinctive label is a fluorescent label.

The skilled artisan will understand that, if one wishes to determine ifa specific genotype is present in a sample, e.g., a “T” in a position inthe TIGR sequence that would be a “G” in the wild-type sequence, oneneed only provide a single labeled ddNTP, in this case ddTTP, with anappropriate extension primer. If the “T” polymorphism is present, thelabeled ddTTP will be incorporated into the 3′ end of the extensionprimer. In contrast, if the wild-type sequence is present, no labeledextension primer will be created. Depending on the polymorphismsselected for analysis, from one to four labeled ddNTPs may be requiredto perform an assay. One may also choose to include all four ddNTPs in areaction for convenience, or so that even wild-type sequences becomelabeled.

As used herein, “primer extension” refers to the enzymatic extension ofthe three-prime (3′) hydroxy group of an extension primer, which is anoligonucleotide that is paired in a duplex to a template nucleic acid.For an example of primer extension as applied to the detection ofpolymorphisms, see Fahy et al., Mutliplex fluorescence-based primerextension method for quantative mutation analysis of mitrochondrial DNAand its diagnostic application for Alzheimer's disease, Nucleic AcidResearch 25:3102-3109, 1997. The extension reaction is catalyzed by aDNA polymerase. By “DNA Polymerase” it is meant a DNA polymerase, or afragment thereof, that is capable of catalyzing the addition of bases toa primer sequence in a sequence-specific fashion. A DNA polymerase canbe an intact DNA polymerase, a mutant DNA polymerase, an active fragmentfrom a DNA polyerase, such as the Klenow fragment of E. coli DNApolymerase, and a DNA polymerase from any species, including but notlimited to thermophilic organisms.

Extension of the 3′ end of the oligonucleotide generates anoligonucleotide having a length greater than the extension primer andhaving a sequence that is the reverse complement of the template nucleicacid. If one of the nucleotides in the added sequence is labeled, thenthe extended oligonucleotide becomes labeled.

Preferably, an extension primer has a nucleotide sequence that binds ina complementary fashion to a portion of a nucleic acid sequence thatencodes or modulates the expression of the TIGR gene, or to thecomplement of such a sequence. Extension primers must be of a lengthsufficient to provide specific binding to the target sequence ofinterest. Such primers comprise an exact complement to the sequence ofinterest for 15 to 75 nucleotides in length, preferably 17 to 50nucleotides in length, and more preferably from 20 to 30 nucleotides inlength. The extension primer sequence has a 3′ terminus that pairs witha nucleotide base that is, in the sample nucleic acid to which theprimer is hybridized, 5′ from the site of one or more bases in thesequence of interest that represent a polymorphism in a gene. Suitableextension primers are described herein, and may be one of the sequencesset forth in SEQ ID NOS:1-4.

In addition to the sequence that ensures hybridization to the targetsite, an extension primer may have additional nucleotides added to the5′ end that need not participate in specific binding. Thus, such primersmay extend for 15 to 75 nucleotides in length, preferably 17 to 50nucleotides in length, and more preferably from 20 to 30 nucleotides inlength, only a subset of which is an exact complement to the sequence ofinterest. In these embodiments, the exact complement may extend for atleast 15 nucleotides, more preferably for at least 17 nucleotides, andmost preferably for at least 20 nucleotides to ensure specifichybridization of the extension primer. Thus, an extension primer maycontain one of the sequences set forth in SEQ ID NOS:1-4 at the 3′ end,with additional nucleotides at the 5′ end which may or may not becomplementary to the TIGR sequence of interest.

In the following diagram of a primer extension reaction, four differentddNTPs, each distinctively labeled, are present in the reaction mixtureas designated by dd(A*1)TP, dd(T*2)TP, dd(C*3)TP and dd(G*4)TP, where*1, *2, *3 and *4 represent different labels. In the diagram, thepolymorphism in the nucleic acid being tested is indicated by anunderlined nucleotide, and the extension primer sequence is italicized.Only one ddNTP, ddTTP, can be added to the 3′ end of the extensionprimer, because thymine (T) is the only base that pairs with adenosine(A). The addition of the dd(T*2)TP to the 3′ of the primer prevents anyfurther primer extension because it is a dideoxy, chain-terminatingddNTP. Thus, the only primer that is 3′ extended is labeled with label*2. Detection of the signal from label *2 indicates that the Apolymorphism is present in the sample:

wild-    5′-CCGGGGTGGTTGGCGAAGGCAGTCCCCTGTGCTGCC-3′ type

labeled ddNTP dd(A*¹)TP        3′-CACCAACCGCTTCCGTCAGTGGA-5′dd(T*²)TP     3′-*² T CACCAACCGCTTCCGTCAGTGGA-5′dd(C*³)TP        3′-CACCAACCGCTTCCGTCAGTGGA-5′dd(G*⁴)TP        3′-CACCAACCGCTTCCGTCAGTGGA-5′

As discussed herein, an amount of nucleic acid sufficient for primerextension can, but need not be, prepared by amplification, e.g., via PCRusing amplification primers. As a non-limiting example, appropriateamplification primers include, but are not limited to, those havingsequences set forth in SEQ ID NOS:5-9.

For each reaction mixture, the amount of the nucleic acid sufficient forprimer extension can be determined by obtaining a sample comprisingnucleic acid and determining the concentration of nucleic acid therein.One skilled in the art will be able to prepare such samples to aconcentration and purity necessary to practice the invention, and toestimate the amount of a specific sample that should be added to aparticular reaction mixture. A failure to detect a signal in the methodof the invention may signify that, among other things, an inadequateamount of nucleic acid has been added to a reaction mixture. Thoseskilled in the art will be able to trouble-shoot failed batch runs andadjust the contents of the reaction mixtures and/or conditions of therun accordingly. Control samples, both positive and negative, can beincluded in the batch runs to confirm that appropriate amounts ofnucleic acid are present.

One or more of steps of the assays described herein, in any combination,are preferably performed in an automated fashion, typically usingrobotics, in order to provide for the processing of a large number ofsamples in a single batch run. Preferred forms of automation willprovide for the preparation and separation of a plurality of labelednucleic acids in small volumes. The term “small volumes” refers tovolumes of liquids less than 2 ml, e.g., any volume from about 0.001picoliters or about 0.001 μl, to any volume about 2 ml, 500 μl, 200 μl,100 μl, 10 μl, 1 μl, 0.1 μl, 0.01 μl, or 0.001 μl.

The set of distinctively labeled oligonucleotides generated by themethods described herein can be separated from each other so that eachis mobilized in a manner that relates to each of their specificpositions in the respective nucleotide sequence, and the detection ofthe distinctive signals generated from the distinctively labeledoligonucleotides occurs during or after the mobilization. Members of theset of distinctively labeled oligonucleotides can be separated from eachother so that each is mobilized by electrophoresis. A preferred form ofelectrophoresis is capillary electrophoresis, but any form ofelectrophoresis that allows for the separation of a plurality of labelednucleic acids in small volumes by automated or semi-automated methodsand devices may be used.

In additional aspects of the present invention, polymorphic forms ofTIGR assayed according to the invention can be used to diagnose subjectssuffering from glaucoma or to identify subjects that are at increasedrisk for developing glaucoma. In preferred embodiments, such subjectsmay also exhibit one or more additional risk factors for glaucoma,including elevated IOP (≧22 mm Hg, more preferably 22-30 mm Hg, mostpreferably 27-30 mm Hg), increased age (≧60 years of age), a familyhistory of glaucoma, diabetes, hypertension, myopia, and the use ofcorticosteroids. In certain embodiments, such subjects exhibit a normalIOP (11-21 mm Hg).

In other aspects, the results from the assays of the invention can beused to initiate or design a regimen of treatment, based on an increasedrisk for developing glaucoma. Such treatment may include one or more ofthe following: surgical or laser treatments, installation of shunts,treatment with miotics (such as pilocarpine, carbachol, physostigmine,demecarium, and isofluorophate), treatment with carbonic anhydraseinhibitors (such as acetazolamide and methazolamide), adrenergicagonists (such as epinephrine, dipivefrine, and α2-specific agonistssuch as apraclonidine), β-blockers (such as betaxolol and metipranolol),prostaglandin analogs, and osmotic diuretics.

In yet other aspects, the present invention relates to one or moreoligonucleotide molecules that are amplification primers and/orextension primers for use in the present invention. Preferably, theextension primers comprise sequences selected from the group consistingof SEQ ID NOS: 1-4, and the amplification primers comprise sequencesselected from the group consisting of SEQ ID NOS: 5-8. Preferably, theoligonucleotide molecules of the present invention are purified, andmost preferably substantially pure molecules.

As used herein, the term “purified” in reference to oligonucleotidesdoes not require absolute purity. Instead, it represents an indicationthat the sequence is relatively more pure than in the naturalenvironment. Such oligonucleotides may be obtained by a number ofmethods including, for example, laboratory synthesis, restriction enzymedigestion or PCR. A “purified” oligonucleotide is preferably at least10% pure. A “substantially purified” oligonucleotide is preferably atleast 50% pure, more preferably at least 75% pure, and most preferablyat least 95% pure.

In further aspects, the present invention also relates to kits forperforming the methods described herein. Preferably, such kits containone or more extension primers in an amount sufficient to perform atleast one assay for determining the presence or absence of a particularpolymorphic form of TIGR in a sample. More preferably, such kits containextension primers in an amount sufficient to perform at least one assayfor determining the presence or absence of at least two, and mostpreferably at least four, different polymorphic forms of TIGR in asample. Preferably, the extension primers comprise sequences selectedfrom the group consisting of SEQ ID NOS: 1-4. In certain embodiments,the kits also contain amplification primers in an amount sufficient toperform a PCR amplification of the polymorphism region(s) of interest inthe assay. Preferably, the amplification primers have sequences selectedfrom the group consisting of SEQ ID NOS: 5-8. In certain otherembodiments, the kits may also contain an instruction manual providinginstructions for use of the extension probes, and amplification probesif present in the kit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 represents the sequence of TIGR exon 3 (SEQ ID NO.: 9).

FIG. 2 represents the sequence of TIGR promoter (SEQ ID NO.: 10).

DETAILED DESCRIPTION OF THE INVENTION

The invention is drawn to assays that are predictive and/or diagnosticfor glaucoma. In particular, the invention provides methods andcompositions for determining the presence and sequence of variantalleles of genes encoding TIGR.

TIGR Polymorphisms

In a normal diploid eukaryote, each gene has 2 loci, i.e., 1 gene copyat the same locus (position) on each of 2 matched chromosomes. Differentversions of a gene can occur at any locus, and these versions are calledalleles. Each allele may be the wild-type (normal) allele or an allelicvariant. Thus, two different versions of a TIGR gene will be present inany particular subject.

By “allelic variant” it is meant a variation in a nucleotide sequence,such as a single nucleotide polymorphism (SNP) or any other variantnucleic acid sequence or structure (e.g., duplications, deletions,inversions, insertions, translocations, etc.) in a gene encoding a genethat alters the activity and/or expression of the gene. Allelic variantsand/or over- or under-express the polypeptide encoded by the gene,and/or express proteins altered activities by virtue of having aminoacid sequences that vary from wildtype sequences.

Often, more than one allelic variant exists and persists in a populationof individuals. By “exist and persist” it is meant that the frequency ofincidence of a rare allele(s) is greater than can be explained byrecurrent mutation alone (i.e., typically greater than 1%). However, thefrequency of any variant allele may vary over time due to such factorsas genetic drift and the like. When 2 or more different alleles of agene are present in a population, the gene or the protein it encodes issaid to be polymorphic. As used herein, a “polymorphism” refers to aspecific form of a gene or protein.

Various forms of the TIGR gene can signal varying risks of developingPOAG, and/or varying severity of the disease. For example, the MT-Imutation can be associated with greater visual field loss, elevated IOP,and refractoriness to conventional medical therapy compared to POAGpatients not exhibiting this polymorphism. In the case of the T377Mmutation, reported glaucoma rates for carriers are 100% by the age of44. Similarly, for the K423E polymorphism, reported glaucoma rates forcarriers are 10% by age 19, 74% between age 20 and 40, and 90% at age 40or older; and for the N480K polymorphism, 25% by age 19, 50% by age 26,75% by age 32, and 95% by age 57. The following table indicates theprevalence of each of these mutations in the population of POAGpatients:

Mutation Prevalence MT-1 17% T377M 0.12 E423K 0.4 N480K 0.81

The following additional allelic variants have been described in theTIGR gene:

-   -   TYR430HIS;    -   GLY357VAL;    -   GLN368TER;    -   PRO370LEU;    -   ILE477SER;    -   PRO370LEU;    -   GLY367ARG;    -   GLN337ARG;    -   ARG46TER; and    -   CYS433ARG.

Screening for TIGR Polymorphisms

The invention can provide rapid and simultaneous screening for largenumbers of samples and/or for several polymorphisms of TIGR. In anexemplary aspect, the assays of the invention are designed to screen for4 polymorphic loci of the TIGR gene; however, the skilled artisan willunderstand that the methods described herein could be expanded toprovide screening for numerous additional TIGR polymorphic loci.

The screening methods described herein are discussed in reference topolymerase chain reaction (“PCR”) amplification of genomic sequences.The skilled artisan will understand that numerous methods are known inthe art for amplification of nucleic acids, and that these methods maybe used either in place of, or together with, the disclosed PCR steps.Nucleic acid amplification methods, such as PCR, isothermal methods,rolling circle methods, etc., are well known to the skilled artisan.See, e.g., Saiki, “Amplification of Genomic DNA” in PCR Protocols, Inniset al., Eds., Academic Press, San Diego, Calif. 1990, pp 13-20; Wharamet al., Nucleic Acids Res. 2001 Jun. 1;29(11):E54-E54; Hafner et al.,Biotechniques 2001 Apr;30(4):852-6, 858, 860 passim; Zhong et al.,Biotechniques 2001 Apr;30(4):852-6, 858, 860 passim.

In embodiments where RNA (e.g., TIGR hnRNA or mRNA) is to be screened,amplification can be carried out using a combined reversetranscription-polymerase chain reaction (RT-PCR) amplification, in whicha single enzyme catalyzes the primer extension both from the initialgenomic RNA template (i.e. reverse transcription) and from the DNAtemplates synthesized in the amplification process.

Nucleic acids, including the TIGR sequence of interest, may be isolatedfrom biological samples through the use of routine methods. Variouscommercial nucleic acid purification kits, such as QIAGEN® BioRobot™ andQIAamp® 96 VIRUS kit are known to the skilled artisan, and used toisolate nucleic acids.

In various embodiments, TIGR coding sequences and/or upstream regulatorysequences are amplified prior to analysis by primer extension methods,preferably using PCR. Suitable forward and reverse primer sequencesshould flank the polymorphism region of interest. Exemplary primers fortwo regions of the TIGR gene are as follows:

Primer Name Primer Sequence TIGRe3F 5′-gcg gtc cca aaa ggg tca gtg tatggt gtg tgg atg cga g-3′ (SEQ ID NO.: 5) (exon 3) TIGRe3R 5′-gcg gtc ccaaaa ggg tca gta agt tgt ccc agg caa aga g-3′ (SEQ ID NO.: 6) (exon 3)TIGRmt1F 5′-gcg gtc cca aaa ggg tca gtg cga ata gag cca taa act ca-3′(SEQ ID NO.: 7) (promoter) TIGRmt1R 5′-gcg gtc cca aaa ggg tca gta tctggg gaa ctc ttc tca g-3′ (SEQ ID NO.: 8) (promoter)

Amplification primer sequences may comprise a 3′ end that ensureshybridization to the target site, and may also include additionalnucleotides added to the 5′ end that need not participate in specificbinding. Thus, such amplification primers may extend for 15 to 75nucleotides in length, preferably 17 to 50 nucleotides in length, andmore preferably from 20 to 30 nucleotides in length, only a subset ofwhich is an exact complement to the target sequence of interest. Inthese embodiments, the exact complement may extend for at least 15nucleotides, more preferably for at least 17 nucleotides, and mostpreferably for at least 20 nucleotides to ensure specific hybridizationof the amplification primer.

Once amplified, the PCR products are treated, e.g., with Shrimp AlkalinePhosphatase (SAP) and Exonuclease I, to remove excess dNTPs and PCRprimers. This is followed by a single nucleotide primer extensionSNaPshot reaction (Lindblad-Toh et al., Large-scale discovery andgenotyping of single-nucleotide polymorphisms in the mouse. NatureGenet. 2000 April;24(4):381-6). In this reaction, an oligonucleotideprimer is designed to have a 3′ end that is one nucleotide 5′ to aspecific point mutation site or to a specific sequence found at aboundary of a mutation that occurs at a larger scale (e.g., aduplication, deletion, inversion, etc.). The primer hybridizes to thePCR amplicon in the presence of fluorescently labeled ddNTPs and a DNApolymerase. The polymerase extends the primer by one nucleotide, addinga single, labeled ddNTP to its 3′ end. Each dideoxynucleotide (e.g.,ddATP, ddGTP, ddCTP, ddTTP, ddUTP, etc.) is differently labeled, e.g.,each is labeled with a different fluorescent colored dye. The primersare tagged with varying lengths of nonspecific polynucleotides (e.g.,poly-GACT) to allow multiplex detection of 5 or more, preferably 10 ormore, and most preferably 100 or more different mutations(polymorphisms) in a single reaction. Excess ddNTPs are removed from thereaction mixture by SAP treatment. The products are fluorescentlylabeled oligonucleotides, each one of which may be detected, for exampleusing an automated DNA sequencer (e.g., ABI PRISM 3100 Genetic Analyzer)based on its size (determined by electrophoretic mobility) and/or itsrespective fluorescent label.

Suitable extension primers for several polymorphism genotypes are asfollows:

Primer Polymorphism Name Primer Sequence MT-1 SNP32 5′-cga ata gag ccataa act caa agt ggt aat aa-3′ (SEQ ID NO.: 1) T377M SNP33Δ 5′-ccg tattct tgg ggt ggc tac a-3′ (SEQ ID NO.: 2) K423E SNP34Δ3 5′-ctc aaa cctggg aga caa aca tcc gt-3′ (SEQ ID NO.: 3) N480K SNP35Δ 5′-gac tgc tataag tac agc agc atg att gac tac aa-3′ (SEQ ID NO.: 4)

EXAMPLES

The following examples serve to illustrate the present invention. Theseexamples are in no way intended to limit the scope of the invention.

Example 1 Preparation of Biological Samples

Biological samples and other specimens are obtained, stored and preparedfor assay using protocols that may vary depending on the type of samplethat is to be used in the assay.

1.1 Obtaining Specimens

Any biological sample containing TIGR nucleic acids may be a suitablesample. For genomic DNA, a convenient sample can be obtained by rinsingthe mouth with water to remove loose particles, followed by scraping thecheek to collect buccal epithelial cells.

Example 2 Instruments and Equipment

The following commercially available instruments and equipment arenon-limiting examples of those that may be used to practice theinvention. Those skilled in the art will be able to determine otherinstruments and equipment that may be used in the methods of theinvention.

2.1 Pipettes

Standard pipettes are used to deliver volumes ranging from 0.5 to 100mL. For volumes less than 1 ml, pipettors such as the P-10, P-20, P-200,P-1000 (Rainin Instruments, LLC) pipettors are used. Pipet tips areselected from Barrier Pipet Tips (Robbins Scientific); Pipet Tips, 20 μland 250 μl (Beckman), and ART (aerosol resistant tips) for P-10, P-20,P-200, P-1000) (Rainin).

2.2 Electrophoresis

Examples of apparatuses that may be useful for electrophoresis andvisualization are an agarose gel electrophoresis apparatus, such as CBSScientific horizontal mini-gel; a power supply having a constant voltageof 200V or better variable power supply for electrophoresis, such as theBioRad Model 200; photodocumentation apparatus, such as the AlphaInnotech Alphalmager or Polaroid DS34 t; and a transilluminator, e.g., aVWR Model LM-20E or equivalent.

2.3 Centrifugation

Centrifugation is carried in BIOMEK® 2000 or Vortex (VWR; G-560)instruments and centrifuges for spinning PCR trays (Sorvall T6000D). The96-well-plate centrifugation system from Qiagen may also be used.Microcentrifuges such as those from Eppendorf are used withMicrocentrifuge tubes (from, e.g., National Scientific, CN065S-GT).

2.4 PCR Containers and Reaction Plates

For DNA amplification (PCR), 2 ml MicroTubes with screw caps (Sarstedt;72.693-005) may be used. A variety of 96-well plates suitable for PCRand other manipulations can be used. In the Examples herein, ABIMicroAmp Optical 96-well Reaction Plates (P/N#N801-0.0560) are used withABI 96-well Plate Septa (P/N#4315933), or Microseal 96-well PCRmicroplates (MJ Research, MSP-9601) are used with Microseal A sealingfilm for microplates (MJ Research, MSA-5001). A 96-place storage systemexemplified by VWR #30128-330, is used to store plates containingsamples between steps in the assay.

2.5 PCR Cycler

A PCR cycler capable of processing 96-well plates is used in theExamples. Exemplary PCT thermal cyclers include the GeneAmp 9600(Perkin-Elmer) or the PTC 200 (MJ Research). The MJR PTC 200 hasfeatures that are desirable regardless of which instrument is used:heating rates of up to 3° C./second, which reduce reaction times, andrapid temperature homogeneity (e.g., ±0.4° C. within 30 seconds at 90°C.). The heating block that is used may be, for example, VWR's HeatBlock (VWR, 13259-007).

2.6 Automated Laboratory Workstation

In order to process a large number of samples, a multipurpose automatedor semi-automated programmable workstation is used (Meldrum, Automationfor Genomics, Part One: Preparation for Sequencing, Genome Research,10:1081-1092, 2000; Meldrum, Automation for Genomics, Part Two:Squencers, Microarrays, and Future Trends, Genome Research,10:1288-1303, 2000). Preferred features of the workstation include theability to rapidly and accurately pipette, dilute and dispense smallvolumes of liquids. The exemplary programable workstation used herein isthe BIOMEK® 2000 (Beckman Coulter, Inc.).

2.7 Capillary Electrophoresis DNA Sequencer

For high throughput of PCR products, an automated capillaryelectrophoresis (CE) system is used in order to separate labeled DNAmolecules in a size-dependent manner, so that signals corresponding toeach nucleotide in a sequence are detected in a sequential fashion. Forreviews of the use of CE in DNA sequencing and polymorphism analysis,see Heller, Electrophoresis 22:629-43, 2001; Dovichi et al., Methods MolBiol 167:225-39, 2001; Mitchelson, Methods Mol Biol 162:3-26, 2001; andDolnik, J Biochem Biophys Methods 41:103-19, 1999. In the Examples, theABI PRISMS® 3100 Genetic Analyzer is used with an ABI PRISM 3100capillary array, 36-cm (P/N#4315931). This provides a multi-colorfluorescence-based DNA analysis system that uses capillaryelectrophoresis with 16 capillaries operating in parallel to separatelabeled PCR products. A CE DNA sequencer/analyzer that operates 96capillaries may be preferable in assays wherein 96-well plates are used.Analyzers with the capacity to process 96 wells include the MegaBACE™1000 DNA Analysis System (Molecular Dynamics, Inc and Amersham PharmaciaBiotech) and the 3700 DNA Analyzer from (Perkin-Elmer Biosystems).

Example 3 Reagents

3.1 Stock Reagents

The following exemplary stock reagents are used and are stable for theindicated times when stored at the indicated temperature/conditions.

3.1.1 Agarose, SeaKem GTG (FMC 50074). Store ambient (18° C.-26° C.),stable for 1 year.

3.1.2 dNTP set, ultrapure, 100 mM solution (Pharmacia 27-2035-01). Storeat −10° C. to −30° C., stable for 1 year.

3.1.3 EDTA, disodium (Sigma E-5134). Store ambient (18° C.-26° C.),stable for 1 year.

3.1.4 Ethidium bromide (Life Technologies 15585-011). Store ambient (18°C.-26° C.), stable for 1 year.

3.1.4 Ficoll (Sigma, Cat. #F2637). Store at 18-25° C., stable for 1year.

3.1.5 Bromophenol Blue (Sigma, cat. #B6131). Store at 18-25° C., stablefor 1 year.

3.1.6 Xylene Cyanol (Kodak, cat. #1B72120). Store at 18-25° C., stablefor 1 year.

3.1.7 0.5 M EDTA, pH 8.0 (Amresco, cat. #E177), Store at 18-25° C.,stable for 1 year.

3.1.8 Taq Extender PCR additive (Stratagene 600148) stored at −20° C.,stable for 1 year.

3.1.9 If a commercially available DNA extraction kit is not used,reagents for the Proteinase K or phenol-chloroform extraction methodshould be prepared as is known in the art.

3.1.10 ABI 3100 POP-4 polymer (P/N4316335), stable for 1 year whenstored at 2 to 10° C.

3.2 Stock Solutions

The following exemplary stock solutions are used and are stable for theindicated times when stored at the indicated temperature/conditions.

3.2.1 Water, molecular biology grade (BioWhittaker 16-001 Y orequivalent) stored ambient (18° C.-26° C.), stable for 1 year.

3.2.2 6× Gel loading dye (no xylene cyanol)

3.2.3 100 mM disodium EDTA pH 8.0

3.2.4 6%-12% (w/v) Ficoll 400

3.2.5 0.25% (w/v) bromophenol blue

3.2.6 10×TBE buffer

3.2.6.1 Prepare as 890 mM Tris Base, 890 mM Boric Acid, and 20 mMDisodium EDTA

3.2.6.2 TBE buffer (Amresco 0658 or equivalent) stored ambient (18°C.-26° C.), stable for 1 year.

3.2.7 ABI 10×Buffer (P/N402824), stored at 2 to 10° C., stable for 1year.

3.2.8 ABI Hi Di Formamide (P/N4311320). Stored at −10° C. or colder,stable for 1 year or until the indicated expiration date.

3.2.10 100×TE buffer (Sigma T-9285 or equivalent) stored ambient (18°C.-26° C.), stable for 1 year.

3.2.11 ABI 5× Sequencing Buffer, PE Applied Biosytems, (P/N4305603),stored at −15° C. to −25° C., stable for 1 year.

3.3 Kits

The following exemplary kits may be used and are stable for theindicated time when stored at the indicated temperature/conditions.

3.3.1 ABI SNAPshot multiplex kit (P/N4323161), stored at −10 to −30° C.,stable for 6 months

3.3.2 HotStarTaq™ PCR Core Kit (Qiagen 203203 or 203205) (HotStarTaq™enzyme, 25 mMg++, M10×buffer & 5×Q Solution), stable for 1 year whenstored at −10° C. to −30° C.

3.4 Enzymes

The following exemplary enzymes may be used and are stable for theindicated time when stored at the indicated temperature/conditions.

3.4.1 Shrimp Alkaline Phosphatase (USB Corporation, P/N70092X), stablefor 6 months when stored at −10 to −3° C.

3.4.2 Exonuclease I (USB Corporation, P/N70073X), stable for 6 monthswhen stored at −10 to −30° C.

3.5 Standards

The following exemplary standards may be used and are stable for theindicated time when stored at the indicated temperature/conditions.

3.5.1 DNA ladder, 50 bp (Pharmacia Biotech 27-4005-01) stable for 1 yearwhen stored at −20° C.

3.5.2 ABI GeneScan-120 LIZ Size Standard (P/N4322362), stable for sixmonths when stored at 2 to 10° C.

3.6 PCR Amplification Primers

Oligonucleotides used as PCR primers were prepared by OperonTechnologies, Inc. (0.05, 0.2 or 1.0 micromole scale synthesis, no HPLCpurification) and stored as 100 μM stocks at −10° C. or colder,conditions under which they are stable for 1 year. Table 2 gives thesequences of PCR primers used in the Examples.

3.7 Primer Extension Primers

Primer extension primers were prepared by Operon Technologies, Inc.(0.05, 0.2 or 1.0 micromole scale synthesis, HPLC purification): storedas 100 μM stocks at −10° C. or colder, stable for 1 year. The sequencesof primers used in the examples are shown in SEQ ID NOS.: 1-4.

3.8 Working Stocks for PCR, Primer Extension, and SAP Treatment

3.8.1 5× Primer Mix for TIGR Duplex PCR is prepared according to thefollowing recipe and is stable for 1 year when stored at −70° C.

Primer (100 μM) Volume [5X] [working] TIGRe3F    384 μl 2 μM 0.400 μMTIGRe3R    384 μl 2 μM 0.400 μM TIGRmt-1F    384 μl 2 μM 0.400 μMTIGRmt-1R    384 μl 2 μM 0.400 μM H2O:   17664 μl Total: 19200.0 μl

3.8.1.2 TIGR PCR Master Mix is prepared according to the followingrecipe.

Components Per Rxn (μL) ×3000 (μL) 10X Qiagen PCR Buffer 2.5 33000 25 mMdNTP mix 0.25  7500 5X primer mix 5.0 15000 25 mM MgCl₂ 1.0  3000 H2O11.0 33000 Total 19.75 59250 μL

3.8.4 SAP+Exo I Cocktail

Combine 5 μl of SAP (1 unit/μl) and 0.2 μl of Exo I (10 unit/μl) in1×SAP buffer to a final volume of 15 μl per reaction. The SAP+ExoICocktail is prepared fresh before each use.

Concentration Volume (μl) for 120 rxns (full plate) SAP  1 unit/μl 600Exo I 10 unit/μl 24 10x SAP buffer 10x 240 Sterile H20 936 Total 1800

3.8.5 Primer Extension Primer Mix is prepared according to the followingrecipe.

Concentration Volume of primer Volume of primer Primer (μM) added (1rxn) added (200 rxns) SNP32 100 0.005 μL  10 μL (MT-1) SNP33Δ 100  0.02μL  40 μL (T377M) SNP34Δ3 100  0.02 μL  40 μL (K423E) SNP35Δ 100  0.02μL  40 μL (N480K) dH2O 0.935 μL 1870 μL Total:  1.0 μL 2000 μL

The mix is prepared in 15 ml sterile conical tubes and dispensed in 1 to1.5 ml aliquots per microcentrifuge tube and stored at −70° C. orcolder.

3.8.6 SNaPshot Primer Extension Master Mix

Combine 2.5 μL of ABI SNaPshot Ready Mix, 2.5 μL of 5× sequencingbuffer, 1 μL of Primer Extension Primer Mix and 1 μl Sterile H₂O to afinal volume of 7 μl per reaction. The Mix is prepared fresh before eachuse, and kept on ice until used.

Reagent Per Well Per Plate SnaPshot Ready Mix 2.5 μL 280 μL 5Xsequencing buffer 2.5 μL 280 μL Extension Primer Mix   1 μL 112 μL DH₂O  1 μL 112 μL Total   7 μL 784 μL

3.8.7 Second SAP Cocktail:

For each reaction, 1 μl of SAP (1 unit/μl) and 1 μl of water arecombined to a final volume of 2 μl. The SAP cocktail is freshly preparedbefore each use.

3.8.8 Loading Mix: Ten (10) μl of Hi-Di Formamide and 0.5 μl GeneScan120 LIZ Size Standard are combined to a final volume of 10.5 μl persample. Lodging Mix is prepared fresh before each use.

Reagent Per Well Per Plate* Hi-Di Formamide   10 μl 1120 μl GeneScan 120LIZ  0.5 μl  56 μl Size Standard Total 10.5 μl 1176 μl

Example 4 Procedure

4.1 Preparation of Sample Trays

PCR master mix is prepared as described above and is used in thereaction. The following table describes a recipe that results in asufficient volume for a full PCR plate (sample tray; 96-wells), andallows for excessive solution to enable pipetting from a trough with an8-channel pipettor into all PCR wells.

Cocktail × Cocktail × 56 (½ 112 (full 1 Rxn plate) plate) Master Mix19.75 μL 1106 μL 2212 μL HotStarTaq  0.25 μL  14 μL  28 μL BuccalSwab/Qiagen  5.0 μL (or — — DNA* 2.0 Qiagen DNA + 3.0 dH₂O) Total   25μL

If Qiagen DNA is used, add 2.0 μL DNA sample +3.0 μL H₂O per reaction;if a DNA sample is extracted with the phenol/chloroform method, itshould be diluted in sterile water to a concentration of 8-16 μg/ml, andadd 5 μL per reaction.

4.2 PCR Reactions

For automated PCR setup on the BIOMEK® 2000 robotic workstation, the PCRtray, a box of Robbins 125 μL pipet tips, a box of 20 μL pipet tips, theQiagen sample tray and the reagent reservoir (trough) are placed at theappropriate positions on the BIOMEK® 2000 work surface. If the PCR orsubsequent steps are set up manually, the same master mixrecipe/digestion recipe is used, and the assay proceeds as describedbelow without the BIOMEK® 2000, and single or multichannel pipettors andtips are used.

The master mix is added to the reagent reservoir. Eight positions at theend of the Qiagen sample tray are left open for controls. The sampletray is briefly spun down in a plate centrifuge outside of the mastermix and template addition area (i.e., in a clean room). The controlsamples (typically, four positive and two negative controls) are placedin the appropriate positions in the sample tray.

The BIOMEK® 2000 station first pipets 20 μl of the master mix into each0.2 ml PCR tray wells, and then adds 5 μl specimen DNA or control. Thewells are tightly sealed with PCR tube caps pr Microseal A film. Thesample tray is briefly (˜5 s) vortexed and spun down for about 30 s in aplate centrifuge at 2,000-6,000 g (1,600 rpm in a Sorvall T6000Dcentrifuge).

The cycling program (below) is started on a thermal cycler such as theMJR PTC 200. When the temperature reaches >85° C., the PCR tray isplaced in the thermal cycler and its lid is sealed.

The cycling parameters are:

Step Temperature Time 1 95° C.   15 min. 2 94° C.   10 sec 3 55° C.0.50° C./sec. Ramp 4 55° C.   10 sec. 5 72° C. 0.30° C./sec. Ramp 6 72°C.   15 sec. 7 94° C. 0.50° C./sec. Ramp 8 [Go to step 2 and repeat for32 cycles*] 9 70° C.   5 min. 10  4° C. Hold *Typically, 32 cycles isoptimal, however 31-33 cycles may be used if the PCR products from 32cycles are less than optimal.

After PCR is complete, the products may be stored refrigerated up to oneweek or frozen (<−10° C.) if a longer storage period is necessary, orthey may be used immediately in the following procedures.

4.3 First SAP and ExoI Digestion

Digestion starts by adding 5 μl of PCR product and 15 μl of the SAP+ExoICocktail. The plate is sealed, vortexed and spun down in the platecentrifuge. The plate is then placed in the MJR PTC 200 thermal cyclerand a cycling program is run using the following parameters.

Step Temperature Time 1 37° C.  2 hr. 2 75° C. 15 min. 3  4° C. Hold

Each step uses rapid (default) ramp to reach desired temperature. TheSAP/ExoI-treated PCR products can be stored at 2-8° C. until use.

4.4 Primer Extension

SNaPshot Primer extension Master Mix is freshly prepared as describedabove, and 7 μl of the master mix is added to 3 μl of digestion productfrom Example 4.3. After addition of the SNaPshot Primer Extension MasterMix, each plate is immediately placed in the thermocycler and the“SNAPSHOT” program is immediately run.

The plate should not be allowed to sit at room temperature more than 30seconds. The plate is sealed, vortexed and spun down in the platecentrifuge. The plate is then placed in the MJR PTC 200 thermal cyclerand a cycling program is run using the following parameters.

Step Temperature Time 1 96° C. 10 sec. 2 50° C.  5 sec. 3 60° C. 30 sec.4 Go to step 1 24 more times. 5  4° C. Hold

Each step uses rapid (default) ramp to reach desired temperature. Thereaction plates are stored at 2-8° C. until use.

4.5 Second SAP Digestion

2 μl of the SAP Cocktail is mixed with 10 μl primer extension productfrom Example 4.4. The plate is sealed and vortexed, and then spun downin the plate centrifuge. The plate is placed in the MJR PTC 200 thermalcycler and a cycling program is run using the following parameters.

Step Temperature Time 1 37° C.  1 hr. 2 75° C. 15 min. 3  4° C. Hold

Each step uses rapid (default) ramp to reach desired temperature. Thedigestion plate is stored at −15° C. or lower until use.

4.6 Electrophoresis on ABI 3100 Genetic Analyzer

SAP-digested samples are prepared according to Example 4.5 for loadingusing a BIOMEK® 2000. The SNaPShot product is diluted 15-fold withwater, and then 2 μl of the diluted product is mixed with 10.5 μl of theLoading Mix. The plate is covered with septa, vortexed and spun down inthe plate centrifuge. The plate is heated at 95° C. for 5 minutes, thenimmediately placed on ice for 3 minutes or until use. The plate is spundown in a plate centrifuge to collect condensation. The plate is thenassembled and loaded onto the ABI3100 Genetic Analyzer.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method, comprising: (a) incubating a reaction mixture comprising:(i) a sample nucleic acid obtained from a biological sample suspected ofcontaining a TIGR nucleic acid sequence, (ii) a nucleic acid polymerase,(iii) one or more extension primers that specifically bind to said TIGRnucleic acid sequence if present, and that, when extended by onenucleotide at the 3′ end, comprise a nucleotide indicative of one ormore preselected polymorphisms in said TIGR nucleic acid sequence, and(iv) one or more labeled ddNTPs, under conditions such that, in thepresence of said TIGR nucleic acid sequence, said extension primer(s)are distinctively labeled by addition of one of said labeled ddNTP(s) tothe 3′-end of said detection primer, to generate a labeled nucleic acidcorresponding to one of said preselected polymorphism(s); and (b)detecting a signal from said labeled nucleic acid, wherein said signalis related to a TIGR genotype present in said sample; wherein saidsample nucleic acid is obtained by amplification of nucleic acid in saidbiological sample; wherein nucleic acid in said biological sample isamplified by a polymerase chain reaction; and wherein nucleic acid insaid sample is amplified using one or more amplification primersequences selected from the group consisting of SEQ ID NOS:5-8.
 2. Amethod, comprising: (a) incubating a reaction mixture comprising: (i) asample nucleic acid obtained from a biological sample suspected ofcontaining a TIGR nucleic acid sequence, (ii) a nucleic acid polymerase,(iii) one or more extension primers that specifically bind to said TIGRnucleic acid sequence if present, and that, when extended by onenucleotide at the 3′ end, comprise a nucleotide indicative of one ormore preselected polymorphisms in said TIGR nucleic acid sequence, and(iv) one or more labeled ddNTPs, under conditions such that, in thepresence of said TIGR nucleic acid sequence, said extension primer(s)are distinctively labeled by addition of one of said labeled ddNTP(s) tothe 3′-end of said detection primer, to generate a labeled nucleic acidcorresponding to one of said preselected polymorphism(s); and (b)detecting a signal from said labeled nucleic acid, wherein said signalis related to a TIGR genotype present in said sample; and wherein saidextension primers consist of an oligonucleotide 17-50 bases in length,comprising at the 3′ end a sequence selected from the group consistingof SEQ ID NOS: 1-4.
 3. The method of claim 2, wherein said samplenucleic acid is obtained by amplification of nucleic acid in saidbiological sample.
 4. The method of claim 2, wherein said nucleic acidin said biological sample is amplified by a polymerase chain reaction.5. The method of claim 1 or 2, wherein step (b) comprises separatingsaid labeled nucleic acid(s) by electrophoresis.
 6. The method of claim5, wherein said electrophoresis is capillary electrophoresis.
 7. Themethod of claim 1 or 2, wherein steps (a) and (b) are performed byautomated means.
 8. The method of claim 1 or 2, wherein said labeledddNTPs are fluorescently labeled.
 9. The method of claim 1 or 2, whereinsaid labeled ddNTPs comprise ddCTP, ddGTP, ddATP and ddTTP, each ofwhich are physically distinguishable from one another.
 10. The method ofclaim 9, wherein said labeled JdNTPs comprise ddCTP, ddGTP, ddATP andddTTP, each comprise a different fluorescent label.
 11. The method ofclaim 1 or 2, wherein said biological sample is a human sample.
 12. Themethod of claim 11, wherein said human sample is obtained by scrapingwithin the buccal cavity.
 13. A method of identifying a subject atincreased risk for developing primary open angle glaucoma, comprisingcorrelating a TIGR genotype of said subject identified by the method ofclaim 1 or 2 to a relative risk of developing primary open angleglaucoma associated with said TIGR genotype.
 14. A method of selecting atreatment regimen for a subject, said method comprising selecting saidtreatment regimen to be compatible with a TIGR genotype of said subjectidentified by the method of claim 1 or 2.